RESEARCH ARTICLE Biological and chemical control and their

RESEARCH ARTICLE

Biological and chemical control and their combined use tocontrol different stages of the Rhizoctonia disease complexon potato through the growing seasonP.S. Wilson1, P.M. Ahvenniemi1, M.J. Lehtonen1, M. Kukkonen2, H. Rita3 & J.P.T. Valkonen1

1 Department of Applied Biology, University of Helsinki, Helsinki, Finland

2 Department of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland

3 Department of Forest Resource Management, University of Helsinki, Helsinki, Finland

Keywords

Biological control; flutolanil; Gliocladium

catenulatum; logit model; Streptomyces

griseoviridis; Trichoderma harzianum.

Correspondence

J.P.T. Valkonen, Plant Pathology Laboratory,

Department of Applied Biology, University of

Helsinki, PO Box 27, FIN 00014, Finland.

Email: [email protected]

Received: 24 June 2007; revised version

accepted: 13 July 2008.

doi:10.1111/j.1744-7348.2008.00292.x

Abstract

Rhizoctonia solani causes stem canker and black scurf diseases on potato and

negatively affects the yield in all potato-growing areas. While seed-borne infec-

tion can be efficiently controlled by dressing with fungicides, few means of

effective control are available against soil-borne infection. In this study, com-

mercially available antagonistic fungi and bacteria, and the combination of

antagonistic Trichoderma harzianum and seed dressing with flutolanil, were

tested for their efficacy in the control of soil-borne infection of R. solani in the

field. Combined use of flutolanil and T. harzianum was found feasible because

even the highest tested concentration of flutolanil [13.0 lg active ingredient

(a.i.) mL21] had little effect on the growth of T. harzianum in vitro, whereas over

100-fold lower concentrations (0.1 lg a.i. mL21) were sufficient to strongly

inhibit the growth of R. solani (EC50 0.045 � 0.0068 lg a.i. mL211). The varia-

bles under focus in plants inoculated with R. solani were the relative stem lesion

index; sprout/stem number; stolon number, weight and incidence of symptoms

on stolons; total yield and the yield of marketable sized tubers and incidence of

black scurf on the marketable-sized tubers. Flutolanil and its combined applica-

tion with T. harzianum reduced the damage to sprouts and severity of stem

canker at the early stages of growth (up to 30 days postplanting). Towards the

end of the growing season, T. harzianum was required to reduce disease sever-

ity. When applied in-furrow alone or in combination with flutolanil-dressed

seed potatoes, T. harzianum increased the proportion of marketable-sized tubers

in yield from 35% to 60% and decreased the incidence of black scurf on prog-

eny tubers from 31% to 11%, which was not achieved using flutolanil alone.

The number and weight of stolons and the yield of plants remained lower in the

inoculated plants than un-inoculated control plants regardless of the method of

control used. The other two antagonists tested, Streptomyces griseoviridis and Glio-

cladium catenulatum, showed no consistent control of R. solani. Taken together,

the results suggest that combining the application of the antagonist T. harzianum

with seed dressing with flutolanil may provide the best protection of the potato

crop against damage caused by R. solani throughout the growing season.

Introduction

The soil-borne fungus, Rhizoctonia solani Kuhn [teleomorph

Thanatephorus cucumeris (Frank) Donk] causes a disease

complex known as stem canker (lesions and necrosis on

subterranean parts of the plant) and black scurf (sclerotia

on the surface of tubers) on potato (Solanum tuberosum L.)

(Carling & Leiner, 1986). In diseased plants, there is a shift

Annals of Applied Biology ISSN 0003-4746

Ann Appl Biol 0 (2008) 1–14 ª 2008 The Authors

Journal compilation ª 2008 Association of Applied Biologists

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A A B 0 2 9 2

Journal Name Manuscript No. 70165 BDispatch: 11.10.08 Journal: AAB CE: Mohana Priya

Author Received: No. of pages: 14 ME: Senthil Jr.

in tuber size distribution towards increased proportion of

small and/or large progeny tubers, and an increase in the

number of malformed tubers, which reduces the market-

able yield (Frank & Leach, 1980; Jager et al., 1991; Jeger

et al., 1996). Yield reductions caused by R. solani occur

wherever potatoes are grown (Banville et al., 1996; Jeger

et al., 1996).

Inoculum of R. solani can be tuber-borne or soil-borne

(Banville et al., 1996). Infection initiated by the tuber-

borne inoculum is efficiently controlled by dressing seed

potatoes with fungicides (Hide & Cayley, 1982; Jeger

et al., 1996). However, infection based on soil-borne

inoculum is poorly controlled by fungicides, especially

when the levels of initial inoculum are high (Weinhold

et al., 1982; Jager & Velvis, 1985; Hide & Read, 1991;

Brewer & Larkin, 2005; Tsror & Peretz-Alon, 2005).

Application of fungicides into soil may have adverse

environmental consequences, and the efficacy of control

depends on soil type (Kataria & Sunder, 1988). There-

fore, the available means for controlling R. solani on

potato are efficient against tuber-borne inoculum,

whereas better approaches for reducing soil-borne infec-

tion are required.

Biological control by antagonistic micro-organisms has

been proposed as a component of integrated pest manage-

ment, where a range of chemical, cultural and biological

treatments are applied to combat soil-borne R. solani

on potato (Jager et al., 1991; Errampalli et al., 2006).

Various antagonistic fungi, including Trichoderma spp.

(Beagle-Ristaino & Papavizas, 1985; Tsror et al., 2001;

Brewer & Larkin, 2005), Verticillium biguttatum Gams

(Jager & Velvis, 1985; Van den Boogert & Luttikholt,

2004), Laetisaria arvalis Burdsall (Murdoch & Leach,

1993) and nonpathogenic Rhizoctonia spp. (Bandy &

Tavantzis, 1990; Escande & Echandi, 1991; Tsror et al.,

2001), have been reported to control the seed- or soil-

borne infection of R. solani on potato in the field. How-

ever, few studies have addressed the compatibility and

level of control achieved by a combination of biological

and chemical control of R. solani. Integrated control

using an antagonist V. biguttatum and reduced rates of

the fungicide pencycuron has been found to be equal to

chemical control at full rate with respect to reducing the

severity of black scurf and, hence, quality losses in yield

(Jager et al., 1991). It has also improved disease control

compared with the use of the antagonist alone when

infection pressure of R. solani is high (Jager & Velvis,

1986). Integrated control may also be more effective in

reducing the amount of sclerotia developing on progeny

tubers than the chemical control alone at a reduced rate

(Van den Boogert et al., 1990). However, in some stud-

ies, integrated control has not improved disease control

compared with its components applied separately (Elad

et al., 1980; Wicks et al., 1996; Schmiedeknecht et al.,

1998).

Our previous studies carried out in a greenhouse have

shown that T. harzianum Rifai applied to soil decreases

the severity of stem canker caused by soil-borne R. solani

(Wilson et al., 2008). Furthermore, the antagonist re-

duced the severity of black scurf on progeny tubers and

decreased the proportion of small tubers, hence improv-

ing the quality of yield. The aim of this study was to

determine whether control of stem canker and black

scurf is attainable with T. harzianum also in the field, and

whether combined application of this antagonist and

a fungicide could improve the levels of control under the

Nordic climatic conditions.

Materials and methods

Preparation of inoculum of R. solani

An isolate of R. solani (isolate R11) was obtained from

a stem canker lesion of the potato cv. Posmo grown in

Lammi, Finland. It belongs to the anastomosis group 3

that predominates on potato in Finland (Lehtonen et al.,

2008) and was used in the previous study for testing

control of stem canker and black scurf under greenhouse

conditions (Wilson et al., 2008). Growth medium for the

inoculum of R. solani was prepared as described (Wilson

et al., 2008). In brief, 250 g of quinoa seed (Chenopodium

quinoa Willd) and 500 g of quartz sand (grade 0.5–1.2

mm; Optiroc Ltd, Finland 2) were moistened with 275 mL

of reverse osmosis water, and the preparation was steri-

lised in a 1-L bottle by autoclaving twice at 121�C for

1 h. Plugs (10 mm in diameter) cut from the edge of

a 5-day-old colony of R. solani grown on potato dextrose

agar (PDA) were added to the growth substrate in the

bottle and incubated in the dark at 22�C for 3 weeks.

The bottle was shaken daily to ensure an even colonisa-

tion of the substrate by the fungus. The inoculum was

dried on a sterile plastic tray in a laminar flow cabinet

for 48 h and stored at 4�C for 24–48 h before use.

Experimental set up

Field experiments were established in 2 years (2004 and

2005) in the experimental field of the University of

Helsinki, Viikki, Helsinki, Finland (60�13#N, 25�1#E).The soil type was clay, with soil organic matter content

of 6–12% and pH 5.8. Potatoes had not been previously

grown in the fields and no R. solani pathogenic on potato

was known to occur, which was further verified during

the study (see Results). Different areas of the field were

used for the experiments in each year to avoid sources of

infection other than the added inoculum.

Biological and chemical control of R. solani on potato P.S. Wilson et al.

2 Ann Appl Biol 0 (2008) 1–14 ª 2008 The Authors


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Certified, healthy seed tubers of cv. Van Gogh and

Rosamunda (free of black scurf) were obtained from the

Finnish Seed Potato Centre Ltd (Tyrnava, Finland). They

were sprouted in daylight in the greenhouse at 20 � 5�Cfor 2 weeks until the thermal heat sum (>5�C) reached

approximately 220�C. The experimental unit or plot

(1.2 m in length; referred to as treatment plot) consisted

of four tubers planted bymachine at 0.30-m intervals. The

plots were located in 13-m-long rows. The space between

the rows was approximately 0.75 m. All treatment plots

were replicated three times and completely randomised

within blocks. Each sampling date comprised a block.

There were a total of nine and seven blocks (i.e. nine

and seven sampling dates, respectively) in the experi-

ments, totalling 378 (nine dates � 14 treatments � three

replicates), and 168 (seven dates � eight treatments �three replicates) experimental plots in 2004 and 2005,

respectively (Table 1).

In 2004, the seed potatoes were planted on 25 May and

in 2005 on 19 May. Soil fertilisation was performed with

an N : P : K = 8:5:19 manufacture (Perunan Y1; Kemira

GrowHow, Finland3 ) at a rate of 865 and 750 kg ha21

in 2004 and 2005, respectively. Weeds were controlled

with a combination of rimsulfuron [7.5 g active ingredient

(a.i.) ha21; Titus WSB; Kemira GrowHow] and metribuzin

(21.0 g a.i. ha21; Senkor WG; Berner, Finland 4) following

the practices undertaken in commercial potato pro-

duction. For control of potato, late blight Tanos (175 g a.i.

both famoxate and cymoxanil ha21; Berner) and Epok

600 EC (80 and 160 g a.i. metalaxyl-M and fluazinam,

respectively, ha21; Berner) were used in 2004 and 2005,

respectively, when necessary.

Sampling was by hand 7, 14, 21, 28, 35, 42, 60, 90 and

120 days postplanting (dpp) in 2004, and 10, 20, 30, 40,

60, 90 and 120 dpp in 2005. Several sampling dates were

included in the experiments to follow the development of

disease and possible effects of the disease control treat-

ments throughout the growing season. However, the

assessment of yield (amount, proportion of marketable-

sized tubers and incidence of black scurf) was only carried

out at final harvest 120 dpp.

Inoculation

Experiment in 2004

Cultivar Van Gogh was used in the experiment. For the

control of R. solani, treatments included (a) dressing

tubers with T. harzianum by dipping the potatoes briefly

in 1% aqueous solution of Trianum-G (isolate T-22,

Table 1 Experimental set-up in 2004 and 2005

Year Treatment (abbreviation)

Distance of Rhizoctonia

solani inoculum from

seed potato (mm)

Number of sampling

dates (blocks)

Number of

replicates

Number of

experimental plots

2004 R. solani (RHIZ) 0, 30, 60, 90 9 3 108

RHIZ + Trichoderma harzianum dip (THDIP) 0, 30, 60, 90 9 3 108

RHIZ + S. griseoviridis dip (MYCO) 30 9 3 27

RHIZ + G. catenulatum dip (PRES) 30 9 3 27

None (CON) — 9 3 27

T. harzianum dip (CON-THDIP) — 9 3 27

S. griseoviridis dip (CON-MYCO) — 9 3 27

G. catenulatum dip (CON-PRES) — 9 3 27

Total 378

2005 R. solani (RHIZ) 0 7 3 21

RHIZ + T. harzianum dip (THDIP) 0 7 3 21

RHIZ + T. harzianum 50 g m21 (TH50) 0 7 3 21

RHIZ + Moncut dip (MON) 0 7 3 21

RHIZ + MON + TH50 0 7 3 21

None (CON) — 7 3 21

T. harzianum dip (CON-THDIP) — 7 3 21

T. harzianum 50 g m21 (CON-TH50) — 7 3 21

Total 168

CON, no inoculation and no disease control treatment; CON-MYCO, no inoculation with R. solani but dressing with Mycostop; CON-PRES, no inocu-

lation with R. solani but dressing with Prestop (CON-PRES); CON-TH50, TH50 without inoculation with R. solani; CON-THDIP, no inoculation with

R. solani but dressing with Trianum-G; MON, dressing tubers with 0.15% aqueous solution of flutolanil; MON-TH50, combination of dressing with

flutolanil and TH50; MYCO, dressing tubers with 0.01% aqueous solution of Streptomyces griseoviridis (Mycostop); PRES, dressing tubers with 1%

aqueous solution of Gliocladium catenulatum (Prestop); RHIZ, inoculation with Rhizoctonia solani but no disease control treatment; TH50, adding

T. harzianum evenly into the furrow at a rate of 50 g m21; THDIP, dressing tubers with T. harzianum by dipping the potatoes briefly in 1% aqueous

solution of Trianum-G.

P.S. Wilson et al. Biological and chemical control of R. solani on potato



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1.5 � 108 spores g21 preparation; Koppert BV, the Nether-

lands5 ) (treatment referred to as THDIP), (b) dressing tubers

with 0.01% aqueous solution of Streptomyces griseoviridis

Anderson et al. [Mycostop, 108–109 colony-forming units

(CFU) g21 preparation; Kemira] (MYCO) or (c) dressing

tubers with 1% aqueous solution of Gliocladium cat-

enulatum Gilm & Abbott (Prestop, 107–109 CFU g21 prepa-

ration; Verdera, Finland) (PRES) (Table 1). Treatments

were performed immediately before planting.

Inoculum of R. solani (5 g) was added by hand on the

seed tuber or as a ring at certain horizontal distances

(30, 60 or 90 mm) from the tuber, as previously de-

scribed (Wilson et al., 2008). In the MYCO and PRES

treatments, inoculum of R. solani was placed at 30 mm

distance from the seed tuber, whereas in THDIP all

aforementioned distances of inoculum were used

(Table 1). Control treatments included (d) inoculation

with R. solani but no disease control treatment (RHIZ);

or no inoculation with R. solani but dressing with (e) Tri-

anum-G (CON-THDIP), (f) Mycostop (CON-MYCO) or

(g) Prestop (CON-PRES) or (h) no inoculation and no

disease control treatment (CON) (Table 1). The treat-

ment plots planted with cv. Van Gogh were separated

from each other by planting two healthy seed tubers of

cv. Rosamunda between them. Tubers of this cultivar

are red-skinned in contrast to the white-skinned tubers

of Van Gogh, which further facilitated separation of

progeny tubers from the adjacent treatment plots. Two

seed tubers of Rosamunda were also placed at the ends

of each row.

Experiment in 2005

The inoculum of R. solani was added evenly in the fur-

rows at a rate of 5 g m21 at planting (treatment RHIZ).

Treatments for the control of R. solani included (a) dress-

ing tubers with 0.15% aqueous solution of flutolanil

(Moncut 40 SC; 449 g a.i. L21; Berner) (MON); (b)

THDIP (as in 2004); (c) adding the antagonist evenly into

the furrow at a rate of 50 g m21 (TH50) and (d) combi-

nation of dressing with flutolanil and TH50 (MON-TH50)

(Table 1). Control treatments included CON and CON-

THDIP, as in 2004, and TH50 without inoculation with

R. solani (CON-TH50). The treatments plots were sepa-

rated from each other as in 2004.

Disease and yield assessment

On each sampling date, seed tubers with unemerged or

emerged sprouts, roots, stolons and stems, depending on

the developmental stage, was collected and washed with

running tap water. Samples were examined immediately

after collection or were stored at 4�C in the dark for

a maximum of 48 h before closer study. Symptoms

such as lesions and death of unemerged sprouts, and

lesions and discolouration of the subterranean parts of

the stem were collectively called ‘stem canker’ for sim-

plicity. The proportion of the sprouts or stem affected by

the symptoms was scored according to Weinhold et al.

(1982), and each potato stem (sprout) was placed in

one of the six disease severity classes accordingly (0%,

1–5%, 6–25%, 26–50%, 51–75% or 76–100% of the

subterranean part of the potato stem affected). For

each plant, disease severity was then expressed as the

Rhizoctonia stem lesion index (RSI) (Weinhold et al.,

1982) by multiplying the number of stems (sprouts) in

each class by the midpoint of the class and dividing

the sum of the values obtained by the total number of

stems (sprouts) (Weinhold et al., 1982). Thus, the possi-

ble maximum RSI-value for each plant was 88 (mid-

point of the class 76–100%) (Weinhold et al., 1982;

Wilson et al., 2007) 6.

The incidence of infected stolons was determined for

each plant at 60, 90 and 120 dpp. At the last sampling

date 120 dpp, the weight and sizes of progeny tubers

were assessed. Tubers were placed in two size classes:

those 40–60 mm in diameter (marketable size for staple

potato) and those outside this range. Black scurf was visu-

ally assessed on the marketable-sized tubers of each plant

using the illustrated key (reference pictures) of Dijst

(1985) based on the following scale: 0 = free of black

scurf, 1 = very lightly, 2 = lightly, 3 = moderately or

4 = heavily infested with black scurf.

Sensitivity of T. harzianum and R. solani to

flutolanil

The sensitivity to flutolanil of the isolates of R. solani

(R11) and T. harzianum (T-22) was assessed as described

for a collection of 119 R. solani isolates (including R11)

in a previous study (Lehtonen et al., 2008). In brief, au-

toclaved and cooled PDA was amended with different

concentrations of flutolanil (0, 0.1, 0.5, 1.0, 3.0, 5.0,

10.0 or 13.0 lg a.i. mL21) and distributed onto Petri

dishes (20 mL aliquots, diameter of the dish 90 mm). A

plug of PDA with mycelia (diameter 7 mm) was cut

from the leading edge of an actively growing colony of

R. solani or T. harzianum and placed on the edge of the

dish with fungicide-amended PDA. The dishes were

sealed with Parafilm and incubated at 20�C in the dark.

Each isolate and fungicide concentration and also the

fungicide-free control dishes were replicated three times.

Colony growth was measured along two straight lines

drawn from the centre of the mycelial plug (angle

between lines 50�) on days 3, 4, 7 and 10 after inocula-

tion. The growth rate (mm day21) was used to estimate




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the effective concentration causing a 50% reduction in

growth rate (EC50) compared with the fungicide-free

control. The EC50-values were calculated from dose–

response curves using nonlinear regression and the SPSS

statistical software package (SPSS Inc., Chicago, IL, USA).

Statistical analyses

The effects of disease control treatments on the severity of

stem canker and black scurf, and on stem and stolon num-

bers, stolon weight and tuber yield were compared by the

analysis of variance (ANOVA) using the GLM procedure of

the SAS statistical software package (SAS Institute Inc.,

Cary, NC, USA). In the 2004 experiment, a two-way

ANOVA comprising the distance of R. solani inoculum

from seed potato (0, 30, 60 or 90 mm) and disease control

treatment (the presence or absence of T. harzianum)

as factors was used. In the 2005 experiment, one-way

ANOVA was used. The least significant differences were

used to separate the means at P = 0.05.

The incidence of black scurf, the proportion of market-

able tubers (40–60 mm in diameter) in the yield and the

incidence of infected stolonswere analysed by logitmodels.

Logit models were preferred to ANOVA because the

response variables were of a quantal nature (i.e. presence

or absence, forwhich the results are reported as percentages

or proportions). In logit models, the effects of explanatory

variables are described using the concept of odds ratio,

which is a relative measure of difference between the two

probabilities compared: [P2/(1 2 P2)]/[P1/(1 2 P1)] (Col-

lett, 1991; Linden et al., 1996; Hiltunen et al., 2005). Sig-

nificance tests and confidence intervals for the single

parameters were based on Wald statistics. The logit analy-

sis was carried out by the procedure GENMOD of the SAS

statistical software package. When necessary, over-

dispersion of the data was allowed by calculating a scale

parameter that was used to adjust the standard errors and

to correct the chi-squared statistics and P values.

Results

The growing season of 2004 was wetter than average.

The mean rainfall in Helsinki in June, July and August

was 110, 207 and 94 mm, respectively, which was

60, 147 and 19 mm higher than the mean of 30 years

(Venalainen et al., 2005). However, the mean temper-

atures in June (13�C), July (17�C) and August (17�C)did not deviate from the mean of 30 years. In 2005, the

temperatures and rainfall were similar to those recorded

over the past 30 years, and the growing season was

regarded as average.

In the following, the results of mainly those treatments

and sampling dates that caused statistically significant

differences in disease or yield parameters are described

in detail. No stem canker, stolon symptoms or black scurf

on progeny tubers were observed in uninoculated control

plants in 2004 and 2005, indicating that no natural soil-

borne inoculum of R. solani capable of causing symptoms

was present in the experimental fields.

Severity of stem canker and influence of infection

on stem number in 2004

In 2004, inoculum of R. solani (5 g) was placed at different

distances from the seed tuber. At 7 dpp, no shoots had

emerged and the RSI (Weinhold et al., 1982) was deter-

mined based on the damage to the unemerged sprouts.

RSI was highest in the treatments where the inoculum

was placed on or close to the seed tuber (distances 0 or

30 mm) and decreased with increasing distance (60 and

90 mm) of inoculum from the seed tuber, as expected

(Henis & Ben-Yephet, 1970; Tomimatsu & Griffin, 1982;

Gilligan & Bailey, 1997; Wilson et al., 2007) (Table 2a).

At 28 dpp, many but not all shoots had emerged regard-

less of the treatment. Differences in RSI were less

pronounced than at 7 dpp, but the severity of stem can-

ker was still higher in plants with the closer distance from

the inoculum (Table 2a). Overall, these results were

explained by the time needed for the growth of R. solani

towards the plants from the longer distance and which

gradual increased the RSI over time.

Initially, at 7 dpp, sprout numberwas little affected by the

distance of inoculum from the seed tuber (Table 2b).

However, at 28 dpp, a lower total number of sprouts and

stems were observed at shorter distances to the inoculum

(Table 2b). At 60 dpp, the situation had reversed. The

plants at closer inoculum distances had a higher number

of sprouts and stems than those at a longer distance (e.g.

18.7 and 8.9 sprouts/stems per plant at an inoculum dis-

tance of 0 and 90 mm, respectively) (Table 2b), and the

same trend was observed at 90 dpp. Furthermore, there

were no longer significant differences in RSI, regardless of

the distance of inoculum. These data were explained by

branching of sprouts whose tip was killed by infection and

compensatory growth of new sprouts (Baker, 1970; Er-

rampalli et al., 2006) 7in treatments where infection began

early, that is, in treatments where the inoculum was

placed close to the seed tuber, which increased the sprout/

stem number per plant. However, the similar RSI-values

could be explained because stems become more resistant

to stem canker after emergence of potato plants (Van Em-

den, 1965). Hence, the new sprouts/stems of the plants in-

fected early, and the stems of plants in treatments where

R. solani was placed at a longer distance from the seed tuber

and reached the stems late, managed to escape stem canker

development more often than the others (Table 2a,b).




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No antagonist had any significant control effect on stem

canker regardless of the distance of inoculum or time post-

inoculation in 2004 (Table 2a).

Combined chemical and biological control

against stem canker in 2005

In 2005, inoculum of R. solani was applied at the same

amount (5 g) but in a different manner than in 2004.

It was placed evenly in the furrow to simplify the ex-

perimental set-up. Severity of stem canker was signifi-

cantly lower in the MON and MON-TH50 treatments

(RSI 3–22) than in RHIZ (RSI 42–73) at 10 and 30 dpp

(Table 3a). At a later time (60 dpp), RSI was lower only

in treatments including T. harzianum compared with

RHIZ: TH50 had the lowest RSI-value, followed by

MON-TH50 and THDIP (Table 3). At this time point, RSI

of the MON treatment no longer showed any difference

to RHIZ (Table 3). Similar trends were observed at

90 dpp, whereas at 120 dpp, no differences in RSI

were observed between the treatments (RSI 24–41)

(Table 3).

Table 2 The effect of biological disease control treatment and the horizontal distance of inoculum of Rhizoctonia solani from the seed tuber on (a) the

Rhizoctonia stem lesion index (RSI)a and (b) the mean number of stems per plant in 2004

Treatment 7 dpp 28 dpp 60 dpp 90 dpp 120 dpp

(a) Mean RSI

Main effectb

R. solani (RHIZ) 44.0 50.5 33.6 34.9 40.4

RHIZ + Trichoderma

harzianum dip (THDIP)

46.9 53.0 32.1 38.7 44.7

SED (d.f. = 16) 7.1 3.0 3.9 4.7 3.8

Main effectb

RHIZ 0 mm 70.3 54.8 27.5 29.7 35.3

RHIZ 30 mm 74.8 58.1 27.3 29.5 35.6

RHIZ 60 mm 36.0 48.7 37.7 39.7 47.0

RHIZ 90 mm 0.6 45.4 38.8 48.4 52.3

SED (d.f. = 16) 10.0 4.3 5.5 6.7 5.4

30 mm distance of inoculum

RHIZ 72.5 53.4 26.1 32.0 37.0

THDIP 77.1 62.8 28.5 27.0 34.2

RHIZ + S. griseoviridis dip (MYCO) 69.2 57.4 29.2 29.7 30.4

RHIZ + G. catenulatum dip (PRES) 65.0 54.2 33.3 26.1 36.0

SED (d.f. = 8) 22.4 4.4 7.1 2.9 9.2

(b) Mean stem number

Main effectb

RHIZ 5.6 9.0 12.4 8.0 3.9

RHIZ + THDIP 5.0 9.5 14.9 7.9 3.8

SED (d.f. = 16) 0.4 0.5 2.9 1.4 0.6

Main effectb

RHIZ 0 mm 5.8 8.4 18.7 8.3 4.0

RHIZ 30 mm 4.6 8.6 14.7 9.5 3.7

RHIZ 60 mm 4.9 9.3 12.4 7.7 4.0

RHIZ 90 mm 5.8 10.8 8.9 6.3 3.8

SED (d.f. = 16) 0.6 0.8 4.0 2.0 0.9


RHIZ 4.9 9.2 12.5 7.3 3.1

THDIP 4.3 8.0 16.8 11.7 4.3

RHIZ + MYCO 5.8 7.4 13.3 7.9 6.7

RHIZ + PRES 4.2 9.2 12.0 11.3 5.1

SED (d.f. = 8) 0.8 0.7 4.1 2.1 1.7

dpp, days postplanting; PRES, dressing tubers with 1% aqueous solution of Gliocladium catenulatum (Prestop); RHIZ, inoculation with Rhizoctonia

solani but no disease control treatment; MYCO, dressing tubers with 0.01% aqueous solution of Streptomyces griseoviridis (Mycostop); SED,

standard error of difference; THDIP, no inoculation with R. solani but dressing with Trianum-G.aScale 0–88, Weinhold et al. (1982).bInteractions of disease control � distance were not significant at P = 0.05.




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The stem numbers first increased and subsequently

decreased over time in response to the attack by R. solani

when TH50 and/or MON were used for control

(Table 3). However, without control (RHIZ) or using

T. harzianum for seed dressing (THDIP), the stem num-

bers remained low over the whole growing season

(Table 3), which was different from 2004 (Table 2). The

data on development of sprout/stem numbers as a func-

tion of time in 2005 are also illustrated in Fig. 1. They

indicate that inoculum pressure was very high, damage

to unemerged sprouts was severe and no compensatory

growth was possible and sprout/stem numbers remained

constantly low without control (RHIZ) or with ineffi-

cient control (THDIP). However, the control treatments

with TH50, MON and the combined used of MON and

TH50 could delay the damage of sprouts albeit not pre-

vent it, which was reflected as compensatory growth

and increase of the sprout/stem numbers temporarily.

Hence, observation of sprout/stem numbers provided

additional information and indicated a difference in the

severity of the disease in the two growing seasons. The

difference could not be revealed based on the RSI values

of plants inoculated with R. solani (RHIZ; no disease con-

trol) because they were similar in the 2 years. In 2004

and 2005, RSI for RHIZ was 51 and 42 at 28–30 dpp

(t = 21.802, d.f. = 13, P = 0.095), and 34 and 38 at

60 dpp (t = 1.094, d.f. = 13, P = 0.294), respectively.

Compatibility of T. harzianum and flutolanil treatments

was supported by results of in vitro tests. The growth

rate of T. harzianum was little affected by any flutolanil

concentration tested (Fig. 2) and its mean EC50 value

was outside the range of the concentrations used

(13.0 lg a.i. mL21). In contrast, the mean EC50-value of

flutolanil for the growth rate of R. solani was

0.045 � 0.0068 (SE) lg a.i. mL21, which was consistent

with results of a previous study (Lehtonen et al., 2007)

8and indicated a markedly higher sensitivity to flutolanil

of R. solani than T. harzianum.

Damage to stolons caused by R. solani

In both years, the mean fresh weight of stolons per plant

was significantly lower in all the treatments inoculated

with R. solani (1–3 g per plant) than in the uninoculated

Table 3 The effect of biological and chemical disease control treatments on (a) the Rhizoctonia stem lesion index (RSI)a and (b) the mean number of

stems per plant in 2005

Treatmentb 10 dpp 30 dpp 60 dpp 90 dpp 120 dpp

(a) Mean RSI

Rhizoctonia solani (RHIZ) 73.0 42.0 38.3 33.6 34.0

RHIZ + Moncut dip (MON) 8.3 21.6 32.3 35.0 38.5

RHIZ + Trichoderma harzianum

dip (THDIP)

NA 40.6 25.2 31.9 41.2

RHIZ + T. harzianum 50 g m21 (TH50) 49.0 41.1 17.2 20.8 24.0

RHIZ + MON + TH50 3.1 18.8 21.2 26.2 27.6

SED (d.f. = 10) 10.0 (d.f. = 7) 6.3 3.1 2.8 5.5

(b) Mean stem number

RHIZ 3.8 6.8 4.6 3.8 1.9

RHIZ + MON 3.7 10.3 12.3 7.5 1.6

RHIZ + THDIP NA 7.8 6.3 6.0 1.9

RHIZ + TH50 4.6 6.5 11.7 7.7 1.2

RHIZ + MON + TH50 4.5 9.2 13.4 6.8 1.4

SED (d.f. = 10) 0.6 (d.f. = 7) 1.1 1.6 1.9 0.5

dpp, days postplanting; NA, data not available; SED, standard error of difference.aPlants were inoculated with R. solani (RHIZ). Seed dressing with Trichoderma harzianum (THDIP), addition of T. harzianum to furrow (TH50), seed

dressing with flutolanil (MON) or both (MON + TH50) were used for disease control.bScale 0–88, Weinhold et al. (1982).

02468

1012141618

0 30 60 90 120

Days post-planting

Mea

n s

tem

nu

mb

er

Figure 1 The effect of biological and chemical control treatments

on the mean number of sprouts/stems per plant at different time

points postplanting in 2005. All data are from plants inoculated with

Rhizoctonia solani, without or with control. d, R. solani, no control

(RHIZ); n, seed dressing with Moncut (MON); :, seed dressing with Tri-

choderma harzianum (THDIP); s application of T. harzianum to furrow

at 50 g m21 (TH50); h, combined chemical and biological control

(MON + TH50). Each data point represents the mean of three replicates.

Bars represent standard deviation.




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control treatments (5–6 g per plant) and was little

affected by the disease control treatments at any time of

observation (for results of 2005, see Table 4a). Similarly,

in 2005, the mean number of stolons per plant was

lower in the treatments inoculated with R. solani (5–12

stolons per plant) than in the uninoculated control treat-

ments (15–22 stolons per plant), regardless of the disease

control treatments or time of observation (Table 4a).

All stolons in all plants inoculated with R. solani con-

tained lesions in 2004, and no differences in the inci-

dence of infected stolons were observed at 60, 90 or

120 dpp, regardless of the distance between the seed

tuber and the applied inoculum and whether or not dis-

ease control was applied. In 2005, at 60 dpp, the inci-

dence of stolon damage ranged from 32 to 78%, but

differences were not significant between treatments

(data not shown). However, observation of symptoms at

90 dpp indicated that the probability of stolons to be

damaged by R. solani was significantly lower in the TH50

treatment (incidence of stolons with symptoms 36%)

compared with MON-TH50 (49%), MON (56%), THDIP

(65%) or no control (RHIZ; 64%). However, at 120 dpp,

the incidence of stolon damage was significantly lower

in the MON treatment (52%) than in MON-TH50

(70%), THDIP (70%), TH50 (80%) or RHIZ (81%)

(Table 4b). Hence, protection of stolons against damage

was mostly not statistically significant with the control

methods used; however, results suggested some positive

effects because the incidence of stolons with symptoms

was never higher in plants treated with MON and/or

TH50 than the plants (RHIZ) in which no control against

R. solani was used.

Yield and size distribution of progeny tubers

In both years, the mean yield per plant was significantly

lower in all treatments inoculated with R. solani (200–600 g

per plant) compared with the treatments without of

R. solani (1000–1400 g per plant) (Tables 5 and 6a). No

disease control treatment had any detectable effect on the

total yield.

In 2004, inoculation of plants with R. solani (RHIZ) re-

sulted in significantly lower proportions (42–48%) of

marketable-sized tubers (diameter 40–60 mm) in the

yield compared with uninoculated plants (CON, CON-

THDIP, CON-MYCO and CON-PRES) (72–75%), regard-

less of the distance (0–90 mm) at which inoculum was

placed from the seed tuber (Table 5). The disease control

treatments tested (THDIP, MYCO and PRES) did not

result in any statistically significant increase in the total

yield or the proportion of marketable-sized yield in

plants inoculated with R. solani; however, the values for

THDIP were the highest for the total and marketable

yields when inoculum was placed at a 30-mm distance

from the seed tuber (Table 5). The biological control

agents per se (CON-THDIP, CON-MYCO and CON-PRES)

had no negative effect on the yield compared with the

uninoculated and untreated plants (CON) (Table 5).

In 2005, the proportion of marketable-sized tubers

could be increased by the disease control. TH50 increased

the proportion of marketable yield to 60% compared with

35% in RHIZ (Table 6a). Similarly, the combined use of

chemical and biological control (MON-TH50) resulted in

a significantly higher proportion of marketable-sized

progeny tubers than RHIZ (Table 6b). With the two

other disease control treatments (THDIP and MON), the

proportion of marketable yield tended to be higher than

with RHIZ (odds ratio >1), but the difference was not

statistically significant (Table 6b). Trichoderma harzianum

had no negative impact on yield because plants treated

with T. harzianum alone (CON-TH50 and CON-THDIP)

produced similar high yields as the untreated and

R. solani-free plants (CON) (Table 6a,b).

Incidence of black scurf

In 2004, the incidence of marketable sized progeny

tubers with black scurf was higher when the inoculum

of R. solani was placed on the seed tuber (incidence

60%), as compared to placement at distances of 60 mm

(incidence 44%; OR 0.48, P = 0.024) or 90 mm (inci-

dence 42%; OR 0.55, P = 0.046) from the seed tuber.

No biological control agent could significantly prevent

formation of black scurf. While the incidence of black

scurf was 46% in RHIZ (no control), it was 49%, 40%

and 32% in the treatments THDIP, PRES and MYCO,

respectively, at 30 mm distance of inoculum from

the seed tuber (the corresponding OR 1.16, P = 0.6245;

OR 0.79, P = 0.4797 and OR 0.54, P = 0.0798,

respectively).

02468

101214161820

0 0.1 0.5 3 101 135

Flutolanil concentration (ug/ml)

Gro

wth

rat

e (m

m/d

ay)

Figure 2 The growth rate (mm per day) of Rhizoctonia solani (isolate

R11, shaded bars) and Trichoderma harzianum (isolate T-22, unshaded

bars) at different concentrations (lg active ingredient mL21) of the

fungicide flutolanil in vitro. Thin bars indicate standard deviation (n = 3).




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In 2005, incidences of progeny tubers with black scurf

were, in general, similar to those in 2004. The highest in-

cidences in 2005 were observed in THDIP (50%), MON

(44%) and RHIZ (31%) which were not statistically dif-

ferent from each other (Table 7a). Low incidences of 11%

and 18% were observed in the MON-TH50 and TH50

treatments, respectively. In pairwise comparisons using

logit analysis, it was found that the incidence of tubers

with black scurf was significantly lower in MON-TH50

than in RHIZ, MON or THDIP (Table 7b). Furthermore,

the incidence of black scurf in TH50 was lower than in

THDIP and MON (Table 7b).

In both years, the severity of black scurfwas only 1.0–1.5

on the scale of Dijst (1985) in all plants inoculated with

R. solani, and the control treatments had no noticeable

impact on it (data not shown).

Discussion

Concomitant control of stem canker, black scurf and other

negative effects on potato yield, that is, an effective control

through the whole growing season, is expected from the

measures applied to protect the crop against R. solani

(Elad et al., 1980; Brewer & Larkin, 2005). In addition,

Table 4 The effect of biological and chemical disease control treatments on (a) the mean stolon weight and number per plant and (b) the incidence of

stolons with symptoms at 90 and 120 days after planting (dpp) in 2005. The incidences of stolons with symptoms were compared using logit analysis

Treatment

Mean stolon weight (g) Mean stolon number

90 dpp* 120 dpp 90 dpp 120 dpp

(a)

Rhizoctonia solani (RHIZ) 1.7 1.6 6.2 9.5

RHIZ + Moncut dip (MON) 1.5 2.7 8.3 11.9

RHIZ + Trichoderma harzianum dip (THDIP) 1.3 1.6 8.3 10.1

RHIZ + T. harzianum 50 g m21 (TH50) 2.0 2.4 8.8 9.9

RHIZ + MON + TH50 1.8 1.8 5.7 8.4

Control (CON) 5.8 5.3 15.4 19.3

CON + THDIP 5.8 5.6 17.6 17.1

CON + TH50 4.8 4.6 22.0 19.4

SED (d.f. = 19) 1.0 1.3 2.1 2.2

Treatment

Incidence of infected

stolons (%) Odds ratioa95% confidence

intervalbP value of

Wald test

(b)

90 dpp

Comparison RHIZ versus

RHIZ 63.9 1 ND ND

RHIZ + THDIP 65.1 1.05 0.37–2.99 0.9279

RHIZ + TH50 36.1 0.22* 0.08–0.62 0.0041

RHIZ + MON 55.9 0.68 0.24–1.91 0.4704

RHIZ + MON + TH50 49.4 0.55 0.17–1.80 0.3262

Comparison RHIZ + TH50 versus

RHIZ + THDIP 65.1 4.76* 1.83–12.40 0.0014

RHIZ + MON 55.9 3.10* 1.21–7.96 0.0185

120 dpp

Comparison RHIZ versus

RHIZ 81.1 1 ND ND

RHIZ + THDIP 70.1 0.96 0.54–1.69 0.8767

RHIZ + TH50 79.7 1.48 0.80–2.72 0.2082

RHIZ + MON 51.8 0.44* 0.26–0.76 0.0029

RHIZ + MON + TH50 69.6 0.93 0.51–1.68 0.8042

Comparison MON versus

RHIZ + THDIP 70.1 2.16* 1.28–3.65 0.0040

RHIZ + TH50 79.7 3.35* 1.90–5.88 <.0001

RHIZ + MON + TH50 69.6 2.10* 1.21–3.64 0.0084

CON, no inoculation and no disease control treatment; MON, dressing tubers with 0.15% aqueous solution of flutolanil; ND, not determined; RHIZ,

inoculation with Rhizoctonia solani but no disease control treatment; adding T. harzianum evenly into the furrow at a rate of 50 g m21; THDIP,

dressing tubers with T. harzianum by dipping the potatoes briefly in 1% aqueous solution of Trianum-G.aOdds ratio, 1 is equivalent to no difference in the comparison; values <1 indicate increased probability in the first term; values >1 indicate

increased probability in the second term.bIf unity (=1) is not within the confidence interval, the comparison shows a significant difference (P < 0.05)*.




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the control should be effective against seed- and soil-

borne infection, but effective means to control the latter

are scarce. When effective, biological control agents

may have a potential to partially replace or extend the

control spectrum of chemicals (Van den Boogert &

Luttikholt, 2004). In this study, we tested whether the

control of soil-borne inoculum of R. solani is attainable

with three biological control agents in the field but

found only one (T. harzianum) to provide significant

control of the disease. Furthermore, the results indicated

that a combination of chemical control with flutolanil

and biological control with T. harzianum could be more

securely effective against the different components of

the Rhizoctonia disease complex than either method

alone. The reason is that T. harzianum seems to exhibit

control effects throughout the growing season. However,

the combined use of flutolanil and T. harzianum tended

to increase the disease control effects also at the early

growth stages, which suggested that the dose of flutola-

nil used was not harmful to T. harzianum. Because little

data were available on flutolanil sensitivity in T. har-

zianum, it was tested in an in vitro assay. Compared with

R. solani, T. harzianum tolerated over 100-fold higher

doses of flutolanil with little effect on growth, which

Table 5 The effect of biological disease control treatment and the hori-

zontal distance of inoculum of Rhizoctonia solani from the seed tuber on

the mean yield (g per plant), and the proportion of marketable sized

progeny tubers (diameter 40–60 mm) at harvest (120 days postplant-

ing) in 2004

Treatment

Mean

yield (g)

Proportion of

marketable-sized

tubers (%)

Main effect

R. solani (RHIZ) 580.0 50.8

RHIZ + Trichoderma

harzianum dip (THDIP)

619.6 53.6

SED (d.f. = 19) 78.3 ND

Main effect

RHIZ 0 mm 336.0 48.4

RHIZ 30 mm 379.3 52.5

RHIZ 60 mm 351.0 41.5

RHIZ 90 mm 538.7 43.5

Uninoculated control

(CON) with and without

T. harzianum (THDIP)

1353.3 72.7

SED (d.f. = 19) 94.2 ND


RHIZ 349.5 47.6

RHIZ + THDIP 409.0 57.9

RHIZ + S. griseoviridis

dip (MYCO)

287.2 33.9

RHIZ + G. catenulatum

dip (PRES)

193.6 43.1

CON 1378.2 74.4

CON + THDIP 1328.3 71.9

CON + MYCO 1253.3 73.2

CON + PRES 1099.9 75.4

SED (d.f. = 15) 60.2 ND

CON, no inoculation and no disease control treatment; MYCO, dress-

ing tubers with 0.01% aqueous solution of Streptomyces griseoviridis

(Mycostop); ND, not determined; PRES, dressing tubers with 1% aque-

ous solution of Gliocladium catenulatum (Prestop); RHIZ, inoculation

with Rhizoctonia solani but no disease control treatment; SED, stan-

dard error of difference; THDIP, dressing tubers with T. harzianum by

dipping the potatoes briefly in 1% aqueous solution of Trianum-G.9

Table 6 The effect of biological and chemical disease control treatments

on (a) the mean yield (g per plant) and the proportion of marketable-sized

progeny tubers (diameter 40–60 mm) at harvest (120 days postplanting)

in 2005. (b) The proportions of marketable tubers were compared using

logit analysis

(a)

TreatmentaMean

yield (g)

Proportion

of marketable

sized tubers (%)

Rhizoctonia solani (RHIZ) 280.4 35.4

RHIZ + Moncut dip (MON) 392.2 48.1

RHIZ + Trichoderma harzianum

dip (THDIP)

225.6 48.8

RHIZ + T. harzianum

50 g m21 (TH50)

495.8 59.7

RHIZ + MON + TH50 388.1 52.1

Control (CON) 990.1 55.1

CON + THDIP 1093.6 59.5

CON + TH50 1184.3 52.4

SED (d.f. = 19) 97.3 ND

(b)

Comparison

of RHIZ versus Odds ratiob95% confidence

intervalc

P value

of Wald

test

RHIZ + THDIP 1.74 0.81–3.73 0.1538

RHIZ + TH50 2.71* 1.39–5.27 0.0034

RHIZ + MON 1.69 0.83–3.43 0.1452

RHIZ + MON + TH50 1.98* 1.04–3.78 0.0373

CON 2.24* 1.28–3.92 0.0045

CON + THDIP 2.69* 1.50–4.80 0.0008

CON + TH50 2.02* 1.15–3.53 0.0140

CON, no inoculation and no disease control treatment; MON, dressing

tubers with 0.15% aqueous solution of flutolanil; ND, not determined;

RHIZ, inoculation with Rhizoctonia solani but no disease control treat-

ment; sed, standard error of difference; TH50, adding T. harzianum

evenly into the furrow at a rate of 50 g m21;THDIP, dressing tubers

with T. harzianum by dipping the potatoes briefly in 1% aqueous solu-

tion of Trianum-G.aPlants were inoculated with R. solani (RHIZ). Seed dressing with

T. harzianum (THDIP), addition of T. harzianum to furrow (TH50), seed

dressing with flutolanil (MON) or both (MON + TH50) were used for

disease control. For comparison, plants were not inoculated (CON)

and some them were treated with THDIP and TH50 as above.bOdds ratio: 1 is equivalent to no difference in the comparison; values <1

indicate increased probability in the first term; values >1 indicate

increased probability in the second term.cIf unity (= 1) is not within the confidence interval, the comparison shows

a significant difference (P < 0.05)*.




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supports the combined use of flutolanil and T. harzianum

against R. solani.

Seed dressing with flutolanil and its combined use with

T. harzianum was effective in reducing the severity of

stem canker, the disease syndrome caused by R. solani

early in the growing season (Hide & Cayley, 1982; Read

et al., 1989; Scholte, 1989). At this growth stage, the

pathogen can damage and kill sprouts, cause additional

branching and increase in the shoot number but also

delay emergence (Baker, 1970; Errampalli et al., 2006).

However, after the emergence of shoots, development of

new stem canker symptoms ceases (Van Emden, 1965).

These dynamics were also observed in the current study,

as explained in results and illustrated in Fig. 1. It was

found that observation of the sprout and stem numbers

at several time points postplanting provided valuable

information about progression of the disease, for which

RSI alone was not sufficient.

Seed dressing with flutolanil had little effect on the con-

trol of the phases of the disease, which affected the quality

of yield, including the proportion of marketable-sized tu-

bers and black scurf-free tubers among them. For control

of these symptoms that develop late in the growing season

(and further away from the seed tuber), application of

T. harzianum was required. These findings are supported

with data from previous studies showing that T. harzi-

anum is able to grow and colonise new stems, stolons

and roots of the potato plant throughout the growing

season, and thus maintain or increase its controlling effi-

cacy (Harman, 2000; Howell, 2003). In contrast, the

chemical fungicide used for seed dressing is effective

only close to the seed tuber and probably diluted and

washed away during the growing season. The current

study is one of the few (Jager et al., 1991), where the

proportion of marketable-sized tubers could be increased

by application a biological control agent in field con-

ditions. The size distribution achieved following treat-

ment with T. harzianum was similar to those of

uninoculated control plants. Previously, T. harzianum

has been found to reduce the proportion of small prog-

eny tubers of plants inoculated with R. solani in a green-

house experiment (Wilson et al., 2008). However, the

treatments could not prevent the significant yield losses

caused by R. solani. Therefore, while the quality of yield

was improved following application of T. harzianum, the

total marketable yield remained lower than in the non-

inoculated plants.

The data of this study suggest that the probability of

stolon infection was reduced by adding T. harzianum in-

furrow or dressing seed potatoes with flutolanil. These

positive effects on the health of stolons were merely

suggestive because they were not consistently

observed. An important aspect of stolon infection and

damage is that it may interfere with tuber develop-

ment. In previous studies, stolon damage was found to

correlate with an altered size distribution of progeny

tubers, by increasing the proportion of small and large

tubers (Cother, 1983; Scholte, 1989). However, in the

present study, while flutolanil seemed to alleviate sto-

lon infection, it could not improve tuber size distribu-

tion, in contrast to what was observed following

treatment with T. harzianum. Furthermore, T.

harzianum decreased the incidence of black scurf on

progeny tubers, which was not observed with applica-

tion of flutolanil alone. These results are consistent

with the ability of T. harzianum to colonise stolons and

roots, and hence maintain its effectiveness throughout

the growing season (Harman, 2000; Howell, 2003).

Differences observed in the progression of disease and

efficiency of disease control in 2004 and 2005 suggested

that the variable weather conditions and differences in

applying the inoculum played a role. In general, drought

may be controlled by irrigation in field experiments, but

the main difference between the two growing seasons of

this study was the abundant rain falling in 2004 that

could not be controlled. There is some information

Table 7 (a) The effect of biological and chemical disease control treat-

ments on the incidence of progeny tubers with black scurf at harvest

(120 days postplanting) in the marketable-sized progeny tubers in 2005.

(b) Logit analysis was used to compare the incidences

(a)

Treatment

Incidence

of black scurf (%)

Rhizoctonia solani (RHIZ) 31.0

RHIZ + Trichoderma harzianum dip (THDIP) 50.0

RHIZ + T. harzianum 50g m21 (TH50) 17.5

RHIZ + Moncut dip (MON) 44.0

RHIZ + MON + TH50 10.5

(b)

Comparison

Odds

ratioa95% confidence

intervalbP-value of

Wald test

RHIZ versus

RHIZ + MON + TH50 0.26* 0.07–0.96 0.0433

RHIZ + THDIP versus

RHIZ + TH50 0.21* 0.06–0.70 0.0111

RHIZ + MON + TH50 0.12* 0.03–0.46 0.0020

RHIZ + MON versus

RHIZ + TH50 0.27* 0.09–0.84 0.0238

RHIZ + MON + TH50 0.15* 0.04–0.55 0.0043

TH50, adding T. harzianum evenly into the furrow at a rate of 50 g

m21;THDIP, dressing tubers with T. harzianum by dipping the potatoes

briefly in 1% aqueous solution of Trianum-G; MON, dressing tubers

with 0.15% aqueous solution of flutolanil; RHIZ, inoculation with Rhi-

zoctonia solani but no disease control treatment.aOdds ratio: 1 is equivalent to no difference in the comparison; values

<1 indicate increased probability in the first term; values >1 indicate

increased probability in the second term.b If unity (=1) is not within the confidence interval, the comparison

shows a significant difference (P < 0.05)*.




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available concerning the effect of moisture on the

growth of some organisms used as antagonists (Wong

& Griffin, 1974; Eastburn & Butler, 1991; Hussain et al.,

2005), but there is little information about the effect of

moisture on biocontrol activity of the agents tested in

this study (Burpee, 1990). Taken together, it is possible

that, in 2004, soil moisture was excessive for the

antagonists tested. However, a larger amount of inocu-

lum was placed close to the seed tuber in 2004 than

2005. These factors may explain the low efficiency of

control against R. solani in 2004. Temperatures did not

significantly differ between the two growing seasons

and were in the range suitable for the antagonists and

R. solani. According to the manufacturers of the bio-

control agents tested, the suitable temperatures for the

growth of T. harzianum is 10–34�C, for S. griseoviridis

15–25�C and for G. catenulatum 10–30�C. This is the

first time that S. griseoviridis and G. catenulatum have

been tested for the control of R. solani on potato. They

are known to effectively control soil-borne fungal

pathogens, such as Fusarium sp. and Pythium sp. on

greenhouse-grown cucumber (Punja & Yip, 2003; Rose

et al., 2003) but had little effect on R. solani in this

study.

Taken together, the results of this study suggest that

T. harzianum can control R. solani in the field. Previously,

similar results have been obtained in Israel (Elad et al.,

1980; Tsror et al., 2001)m where the climate is much

warmer and therefore the growth conditions are differ-

ent from those of this study carried out in Northern Eu-

rope. The inoculum source (seed or soil) must be taken

into account when planning control strategies against

R. solani on potato (Tsror & Peretz-Alon, 2005). This

study concentrated on soil-borne inoculum of R. solani,

against which chemical control is considered less effec-

tive than against the seed-borne inoculum. Using

healthy seed and adding the inoculum to soil free of nat-

ural soil-borne inoculum of R. solani allowed equalising

the infection pressure in treatment plots, whereas most

of the previous studies carried out in the field have

relied on natural and possibly spatially variable infesta-

tion of the soil. This study is also one of the very few to

report control of R. solani using seed dressing with a fun-

gicide combined with an application of a biological con-

trol agent. In the majority of studies, fungicides have

been applied directly into soil and their effect compared

with those of the biological control agents (Elad et al.,

1980; Beagle-Ristaino & Papavizas, 1985; Jager & Velvis,

1986; Jager et al., 1991; Virgen-Calleros et al., 2000; Van

den Boogert & Luttikholt, 2004). The data obtained in

this study suggest that the combined application of an

effective antagonist and seed dressing with a fungicide

might provide the most effective means of controlling

the different stages of disease and types of damage to the

yield caused by R. solani over the whole growing season.

Acknowledgements

We thank M. German-Kinnari and E. Ketola for assis-

tance during fieldwork and L. Hiltunen for helpful dis-

cussions on data analysis. This work was funded by the

Ministry of Agriculture and Forestry, Finland (grant

4655/501/2003) and the following Finnish companies

and organisations: Berner, Chips, Finnamyl, Finnish

Horticultural Products Society, Finnish Seed Potato Cen-

tre, HG Vilper, Jepuan Peruna, Jarviseudun Peruna,

Kemira GrowHow, Kesko, Krafts Food, MTT AgriFood

Finland North Ostrobothnian Research Station, Northern

Seed Potato, Potwell, ProAgria Association of Rural

Advisory Centeres, ProAgria Rural Advisory Centre of

Oulu, Ravintoraisio-Ruokaperuna , Ruokakesko, Saarioi-

nen and Solanum.

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RESEARCH ARTICLE Biological and chemical control and their

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Transcript of RESEARCH ARTICLE Biological and chemical control and their